The present invention relates to methods for promoting tissue repair, including, but not limited to, wound healing. In the practice of treatment for tissue repair, compositions of complexes of heat shock/stress protein (hsps) including, but not limited to, gp96, hsp90, and hsp70, either alone or in combination with each other, noncovalently bound to antigenic molecules, are used to stimulate cells involved in the normal healing process. Alternatively, compositions containing uncomplexed stress proteins (i.e., free of antigenic molecules) are used to stimulate cells involved in the normal healing process.
2.1. Process of Tissue Repair
The mechanisms of tissue repair involve the interaction of elements of both the coagulation cascade and the immune system. The process of tissue repair is divided into three phases: (1) inflammatory; (2) proliferative; and (3) remodelling. Although these phases are defined as distinct events, they occur as a continuum. The point at which tissue repair begins and ends is based largely upon macroscopic examination.
During the first phase of tissue repair, an acute inflammatory response with cellular migration occurs. Neutrophils predominate for the first 24-48 hours; macrophages become active by the third day. The neutrophils and macrophages phagocytose and digest pathologic organisms and tissue debris (Barbul et al., 1995, Wound Healing for the Otolaryngologist-Head and Neck Surg. 28:955-968).
Macrophage function is crucial in the process of tissue repair. In addition to decontaminating the wound, macrophages synthesize and secrete growth factors such as platelet derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor-alpha (TGF-xcex1), and TGF-xcex2 which promote cell migration and proliferation and matrix formation (e.g., chemoattraction of fibroblasts into the wound edge).
Studies have identified a population of blood-borne cells, termed fibrocytes, that rapidly enter sites of tissue injury (Chesney et al., 1997, Proc. Natl. Acad. Sci. USA 94:6307-6312; Chesney and Bucala, 1997, Biochem. Soc. Transactions 25:520-524). Fibrocytes produce growth factors such as PDGF, FGF, TGF-xcex21; cytokines such as IL-1xcex2 and TNF-xcex1; and matrix components.
Macrophage stimulating protein (MSP), a chemoattractant for peritoneal macrophages, has been shown to induce proliferation and migration of keratinocytes (Wang et al., 1996, Exp. Cancer Res. 226:39-46). Proliferation and differentiation of keratinocytes is important for normal skin function, wound healing, and tumor growth. Id. Thus, MSP may have implications for tissue repair of cutaneous wounds. Stimulation of macrophage function has also been demonstrated to have a beneficial effect on bowel anastomoses (Compton et al., 1996, Amer. Surg. 62:14-18).
The proliferative phase of tissue repair, typically lasting for 4-6 weeks, is characterized by fibroblast migration and neocapillary growth (angiogenesis). The fibroblasts synthesize collagen (as protocollagen, which is then hydroxylated to collagen). Capillary buds, originating in the venules at the edges of the wound, grow across the wound and supply nutrients and oxygen. Newly formed epithelial cells migrate onto the wound surface from the wound margins, resulting in the formation of a well-developed granulation tissue.
During the remodelling stage, cellular activity in the wound decreases. However, the wound continues to gain strength due to collagen cross-linking, remodelling and contraction. Studies have found that when cultured with specific antigen, fibrocytes function as antigen-presenting cells to specifically prime T lymphocytes (Chesney et al., 1997, Proc. Natl. Acad. Sci. USA 94:6307-6312; Chesney and Bucala, 1997, Biochem. Soc. Transactions 25:520-524). Studies suggest an indirect role for T lymphocytes in the wound healing process (administration of agents that enhance T lymphocyte function leads to increases in collagen deposition and wound strength) (Barbul et al., 1995, Wound Healing for the Otolaryngologist-Head and Neck Surg. 28:955968). Myofibroblasts exert a contractile force resulting in wound contraction, thereby decreasing the surface area of the wound, thus helping the wound to close.
2.2. Heat Shock Proteins
Hsps act as molecular chaperones and have been implicated in many processes, including: (1) promotion of folding and unfolding of nascent proteins; (2) participation and activation of a multi-enzymatic complex; (3) formation of complexes with hormone receptor, leading to activation; (4) intra- and intercompartmental transport of proteins; and (5) participation of folding of immunoglobulins (BiP).
Tissue repair encompasses healing of tissues disrupted by trauma (e.g., by surgery or injury) as well as disruption of tissues due to diseases including, but not limited to, atherosclerosis, and multiple sclerosis. Hsps are important for the maintenance of cell integrity during normal growth as well as during pathophysiological conditions (Vigh et al., 1997, Nature Medicine 3(10):1150-1154). Tissue injury, whether caused by surgery, trauma or disease, results in the induction of heat shock/stress proteins (hsps).
Levels of hsp 70 expression have been studied in wound healing. Oberringer et al. has demonstrated a correlation between well-healing wounds and overexpression of hsp 70, whereas chronic wounds exhibited weak or a complete lack of expression. (Oberringer et al., 1995, Biochem. Biophys. Res. Comm. 24(3):1009-1014). Zhu et al. has shown increased expression of hsp 70 in rapidly growing cells, such as those of lesion-prone areas of the vasculature, which may allow proliferating cells an increased chance of survival (Zhu et al., 1996, Arteriosclerosis, Thrombosis, and Vasc. Biol. 16(9) 1104-1111).
Oxidized low density lipoprotein (OxLDL), a causal factor in atherosclerosis, has been shown to induce expression of heat shock proteins (Zhu et al., 1996, Arteriosclerosis, Thrombosis, and Vasc. Biol. 16(9) 1104-1111). In multiple sclerosis, a demyelinating disease, remyelinization is achieved by the proliferation of oligodendrocytes. Studies have shown that heat shock proteins are up-regulated in astrocytes and oligodendrocytes during the course of multiple sclerosis (for review see, Boccaccio and Steinman, 1996, J. Neurosci. Res. 45:647-654).
There is a great need for compositions that promote tissue repair.
Citation or identification of any reference herein shall not be construed as an admission that such reference is available as prior art to the present invention.
The present invention relates to compositions and methods for promoting tissue repair. Methods for treatment comprise administering heat shock proteins (hsps), including but not limited to gp96, hsp90, hsp70, and combinations thereof. The hsps of the present invention may be noncovalently bound to antigenic molecules. The effectiveness of the hsp administration is not dependent on the organ or tissue type or cell from which the hsp was obtained. Accordingly, the treatment regimens disclosed are useful for the repair of a variety of tissues.
Particular compositions of the invention and their properties are described in the sections and subsections which follow. Doses of hsp administered for promoting tissue repair are provided. In addition, the invention provides pharmaceutical formulations for administration of the compositions in appropriate dosages. The invention also provides routes of administration of the compositions used for stimulation of tissue repair.
The examples presented in Section 6, below, demonstrate the use of compositions comprising gp96-peptide complexes in the stimulation of wound healing in a mouse model.